In the past years sophisticated drug depot systems for controlled delivery of substances have been developed, for example to achieve constant drug levels in plasma during therapy. These systems have the advantage of reducing toxic side effects so that the number of drug administrations can be decreased, while at the same time improving cellular uptake and bioavailability. Especially colloidal micro- and nanoparticulate carriers have been extensively investigated as a platform for controlled drug delivery. There is also an ongoing quest to design nano- or microparticles which facilitate controlled release of substances other than pharmaceutical compounds. In general, the material employed as carrier for controlled and sustained release of a substance should offer control of structure, morphology and function, while also exhibiting good mechanical stability.
For example, biodegradable and biocompatible polymers are preferred because of their ability to retain their properties for a limited period of time before gradually decomposing into soluble nontoxic degradation products which can be excreted from the body. Many synthetic (aliphatic polyesters, polyglycolic acid (PGA), polylactid acid (PLA), etc.) and natural (polysaccharides, chitin, chitosan, proteins) polymers have been employed to produce degradable vehicles for encapsulation, incorporation or binding of active compounds [Freiberg, S., Zhu, X. X. Polymer microspheres for controlled drug release. International Journal of Pharmaceutics 2004; 282(1-2):1-18].
While synthetic polymers potentially posses the feature of sustained release of the encapsulated therapeutic agent from a period of days up to several months, they typically demand organic solvents or relatively harsh formulation conditions during processing with potentially limited biocompatibility because of remaining toxic solvents and acidic degradation products.
A further advance in the art was to consider natural polymers which have the advantage of being biocompatible. However, most biopolymers known at present have a major drawback, namely that they resolubilize rapidly in aqueous environment due to their hydrophilic nature, thus resulting in fast drug release profiles. In order to circumvent this problem, chemical cross-linking procedures have been considered. Unfortunately, the presence of residual cross-linking agents can lead to toxic side effects. In addition, undesirable reactions between the drug and the cross-linker could result in the formation of either toxic or inactivated derivatives.
The use of hydrophobic biopolymers as carriers for sustained drug release has also been investigated in the art. For example, silk proteins have been considered as being suitable biopolymers. In particular, silk proteins from spiders and insects, especially Bombyx mori fibroin, have been tested for their ability to deliver drugs and other substances.
For example, silk microspheres consisting of silkworm fibroin for encapsulation and controlled release of a model protein drug has been described in the art. These silk fibroin microspheres with diameters of several microns are obtained by a method using lipid vesicles as a template [Wang, X., Silk microspheres for encapsulation and controlled release. Journal of Controlled Release 2007; 117(3): 360-370].
Larger silk fibroin particles with diameters ranging from 100 to 440 μm and improved loading efficiencies have also been described in the art. However, the preparation techniques for producing these particles are highly sophisticated and lack scalability [Wenk, E., Silk fibroin spheres as a platform for controlled drug delivery. Journal of controlled release 2008; 132(1):26-34].
WO 2007/014755 describes a method of producing nano- and microscapsules consisting of spider silk proteins. These capsules with sizes of several microns are composed of an outer spider silk protein shell and can generally be filled with substances such as proteins or chemical reactants. The microcapsules are formed by the encapsulation of emulsion droplets resulting in hollow spider silk protein shells.
WO 2007/0829223 relates to the use of protein microbeads in cosmetics. In particular, this international patent application describes protein microbeads composed of synthetic spider silk proteins for delivery of cosmetic substances [Hümmerich, D., Primary structure elements of spider dragline silks and their contribution to protein solubility. Biochemistry 2004 Oct. 26; 43(42): 13604-13612]. Similarly, WO 2007/082923 describes the use of protein microbeads for formulating poorly water-soluble effect substances. In both patent applications, the water-insoluble effect substances can be either associated with or encapsulated in the protein microbeads. The association of the substances to these beads is mainly due to hydrophobic interactions. This encapsulation strategy has the basic disadvantage that the loaded substances are only released upon proteolysis of the protein microbeads by the activity of proteases which makes a constant and controlled release difficult. A further problem is that this system is only suitable for the formulation of mainly water-insoluble substances.
Hence, there is a strong need in the field to provide a novel method of producing micro- or submicroparticles with improved qualities. In particular, there is still an ongoing quest to produce nano-scaled particles which are biocompatible and biodegradable as well as being stable carriers for small and water soluble compounds. There is also a need to provide a suitable method of loading silk particles, e.g. spider silk particles, effectively with a compound of interest. The silk particles, e.g. spider silk particles, should also be capable of releasing the loaded compound controllably and sustainably.
Accordingly, it is an object of the present invention to provide a novel and simple drug delivery system which takes into account all of the above criteria. The present invention, therefore, provides a novel method of producing silk particles, preferably spider silk particles, loaded with a compound. More particularly, the method comprises the steps of providing silk particles, preferably spider silk particles, comprising one or more silk polypeptides, preferably spider silk polypeptides, comprising at least two identical repetitive units, and incubating said silk particles, preferably spider silk particles, with at least one compound, wherein the compound is water-soluble and has a molecular weight of between about 50 Da and about 20 kDa.
Surprisingly, one major advantage of the silk carrier system according to the invention is that these particles can be produced and loaded within an all-aqueous system and under ambient condition. This is particularly important with regard to the loading of labile compounds as well as to the overall biocompability of the product. The silk particles, e.g. spider silk particles, of the invention have revealed unexpected loading efficiencies for small and water-soluble compounds. Surprisingly, the silk particles, e.g. spider silk particles, obtained by the method according to the invention have further demonstrated a most favourable release profile, rendering them eminently suitable for controlled and sustained delivery of a compound. The produced silk particles, e.g. spider silk particles, are, therefore, very well suited for delivery of pharmaceutical and cosmetic compounds. Due to their colloidal stability and biocompability under physiological conditions, the loaded silk particles, e.g. spider silk particles, according to the invention are especially suitable for in vivo applications.